Characterization of HLA DR3/DQ2 transgenic mice: a potential humanized animal model for autoimmune disease studies

0014-2980/03/0101-172$17.50+.50/0 © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Characterization of HLA DR3/DQ2 transgenic mice:a potential humanized animal model forautoimmune disease studies

Dan Chen1, Roanna Ueda1, Fiona Harding1, Namrata Patil1, Yifan Mao1, CaroleKurahara2, Gerard Platenburg3 and Manley Huang1

1 Genencor International Inc., Palo Alto, USA2 Amgen Inc., Amgen Center, Thousand Oaks, USA3 Pharming Group, B.V., Niels Bohrweg, Leiden, The Netherlands

Linkage studies indicate close associations of certain HLA alleles with autoimmune dis-eases. To better understand how specific HLA alleles are related to disease pathogenesis,we have generated an HLA DR3/DQ2 transgenic mouse utilizing a 550-kb yeast artificialchromosome (YAC) construct containing the complete DR § , DR g 1, DR g 3, DQ § , and DQ gregions. The transgenic mouse (4D1/C2D) in an I-A g o background appears healthy with nosigns of autoimmune diseases. Lymphoid tissues as well as CD4+ T cells develop normally.Characterization of the transgene expression demonstrates that ˚ 90% of B cells expresshigh levels of DR3 and 50–70% of B cells express DQ2. CD11c+ dendritic cells express highlevels of DR and DQ. Approximately 12–18% of resting T cells are positive for DR expres-sion, and further up-regulation to 40–50% expression is seen upon activation with anti-CD3/anti-CD28 mAb. These results suggest that the transgenic construct confers a high fidelity tothe normal human temporal and spatial expression profile. Analysis of T cell receptor reper-toire in transgenic mice confirms that DR3/DQ2 are able to mediate thymic selection. Fur-thermore, transgenic mice respond to a DR3-restricted antigen, demonstrating antigen pro-cessing and presentation by antigen-presenting cells (APC). Purified T cells from ovalbumin(OVA)-immunized 4D1 mice respond to human APC co-cultured with OVA, suggestingappropriate antigen/DR3 or DQ2 recognition by murine T cells. Immunoglobulin isotypeswitching is also observed, indicating functional T-B cognate interactions. Thus, the DR3/DQ2 transgenic mouse has normal lymphoid development and functionality that are medi-ated by HLA transgenes and can be used to investigate HLA-associated immunologicalquestions.

Key words: Transgenic / Mouse / HLA / Autoimmunity

Received 29/7/02Revised 5/11/02Accepted 25/11/02

[I 23361]

Abbreviations: YAC: Yeast artificial chromosome MFI:Mean fluorescence intensity

1 Introduction

The etiopathology of autoimmunity is variably influencedby environmental factors, genetic background andimmune regulation. The polygenic nature of autoimmunedisease has led to a significant effort to identify disease-associated loci and genes through genetic mapping. Thebest correlation thus far is to the host’s human leukocyteantigen (HLA) loci. The most prevalent autoimmune dis-eases, including rheumatoid arthritis, Graves’ disease,insulin-dependent diabetes, Celiac disease and multiplesclerosis, have close genetic linkages to a limited set of

HLA class II DR or DQ (or both) alleles [1, 2]. In compari-son to the high degree of HLA allele polymorphism, thelimited number of disease-associated alleles providesthe basis for a more focused study on understanding theinfluence of genetic predisposition and pathogenesis atthe molecular and structural level.

The well-defined function of class II molecules is to pre-sent antigens. Preferential binding and presentation ofdisease-inducing self peptide has been implicated as apotential mechanism to shape T cell repertoire, bias Teffector function and influence autoimmunity. Recentstructural studies pinpoint the amino acid residue andstructural similarity in antigen-binding clefts betweendiabetes predisposing alleles of DQ8 in human and I-Ag7

in mouse, and suggest a correlation between betterbinding fit of autoantigen to these disease-prone MHCalleles as compared to non-disease associated alleles

172 D. Chen et al. Eur. J. Immunol. 2003. 33: 172–182

Fig. 1. HLA DQ2/DR3 transgene. A 550-kb YAC clone (in thebracket) from a partial human chromosome that includesTAP and LMP genes as well as the HLA DQ2 and DR3 lociwas used for making transgenic mouse.

[3]. A variety of HLA DR and DQ transgenic mice havebeen developed to evaluate the role of disease-associated alleles in: (1) processing and presentation ofautoantigens, (2) shaping of the T cell repertoire, and (3)influencing T cell effector function leading to autoim-mune pathology. The transgenes have been expressedusing the murine I-E § gene promoter [4–7], as humangenomic clones [8–12] or as hybrids between human andmurine domains [4, 5]. Many of these lines have beencrossed onto mice with a targeted mutation in mouseMHC class I or II genes. In correlation with the diseaseassociation, expression of HLA transgenes renders nor-mally resistant mouse strains susceptible to diseaseonset and affects disease progression. Some transgenicmice develop early signs of disease in the absence ofany immunological manipulation. For instance, sponta-neous development of insulitis is found in DQ8/DR3transgenic mice in a C57BL/6 background [13], and thedevelopment of spondyloarthropathies in HLA B27transgenic rats [14]. Progress has also been made utiliz-ing these animals for some pathogenesis studies[15–18].

The DR3 and DQ2 alleles are found in association with anumber of autoimmune disorders [19, 20]. In an attemptto address the immunological nature of these moleculesin disease susceptibility and modulation, we generated aDR3/DRw52/DQ2 transgenic mouse in the MHC class III-A g –/– background. A 550-kb yeast artificial chromo-some (YAC) clone spanning the human TAP1 throughDR § genes [21] was used to provide a significant portionof linked genes of the class II locus under the control ofhuman regulatory sequences. This strategy has beendemonstrated to be effective at expressing human trans-genes in a copy number-dependent and position-independent manner [22, 23]. Here, we report pheno-typic characterization of the immune system and func-tional assessment of cellular and humoral immunity inthe DR3/DQ2 transgenic mice.

2 Results

2.1 Transgenic mice develop normally and havenormal numbers of CD4+ T cells

To ensure specific recognition of human HLA class II bymurine CD4+ T cells, we crossed the transgenic mouseto the MHC I-A g –/– mouse [24]. The resulting transgenicmice homozygous for I-A g –/– were named 4D1/C2D.

These animals showed no gross developmental andbehavioral abnormalities and no gross pathological signsin various tissues. This is in contrast to other MHC trans-genic mice with high copy numbers of the transgene.

Those animals developed inflammatory disease withprogressive B cell deficiency, abnormal extramedullarygranulopoiesis and increased susceptibility to infection[25]. In 4D1/C2D mice, the thymus, spleen and LNappeared normal in size and cell number. Hematoxylinand eosin-stained tissue sections (Fig. 2A), displayed anormal cellular architecture of the cortex and medulla inthe thymus and normal red and white pulps in the spleen.Primary follicles and periarteriolar lymphoid sheathswere detected in the white pulp and were separated fromthe red pulp by the marginal zone. B cell follicles and theparacortical T cell region in the LN appeared indistin-guishable from control mice. More detailed analyses ofcell types revealed that the number of CD4+ cells and theratio of CD4 to CD8 T cells in the thymus, spleen and LNwere similar to control animals (Fig. 2B). Collectively, thedata indicate that the DR/DQ transgene products actsimilarly to endogenous MHC class II and are able toinstruct thymic development as well as maintain T cellhomeostasis in the periphery.

2.2 Expression profile of HLA DR3 and DQ2molecules in 4D1/C2D mice

LN cells of 4D1/C2D and B6 mice were examined for theexpression of DR and DQ by flow cytometry. Our resultsshow that ˚ 98% of LN B cells expressed high levels ofDR and 60–80% expressed DQ (Fig. 3A). In comparison,100% of human B cells were positive for DR and DQ(Fig. 3A). Examination of T cells showed that the overallpopulation shifted to the DR+ direction with an approxi-mately threefold increase in the mean fluorescenceintensity (MFI). Among them, 12–18% of CD3+ cellsexpressed relatively high levels of DR. In contrast, only2–6% of T cells expressed DQ, the majority of the cellsremained negative. In comparison, human T cellsshowed similar percentages of DR- and DQ-expressingcells with higher levels of DR and DQ expression in bothT and B cells when quantified by the MFI (Fig. 3A). The

Eur. J. Immunol. 2003. 33: 172–182 Development and regulation of immune system in DR3/DQ2 transgenic mice 173

Fig. 2. 4D1/C2D mice have normal lymphoid organ and CD4 T cell development. (A) Hematoxylin and eosin staining of tissuesections of thymus, spleen and LN from B6 and 4D1/C2D mice. (B) The presence of CD4 and CD8 T cells were analyzed in thesetissues and numbers in each quadrant represent the percentage of each population.

DR- and DQ-positive T cells in transgenic mice includedboth CD4+ and CD8+ cells. Additionally, most DR-positive T cells were among the CD44+ memory popula-tion (Fig. 3B), suggesting that DR expression may beacquired during the transition from naı̈ve to memory phe-notype.

Next, the DR and DQ expression in dendritic cells (DC)was examined. Fig. 4A shows that, whereas nearly all theperitoneal CD11c+ cells from 4D1/C2D mice were posi-tive for DR, the levels of DQ expression was much lower.Similar HLA-expressing DC were also found in thespleen and LN (data not shown). As a comparison,human mature DC generated by in vitro culture were alsoanalyzed in parallel and robust expression of both DRand DQ was observed (Fig. 4A).

To address whether the expression of transgenic HLA onDC correlates with their maturity, we compared DR andDQ expression in CD11c+ cells from freshly isolated bonemarrow cells, immature DC from GM-CSF-cultured bonemarrow cells, and mature DC from GM-CSF + LPS-stimulated bone marrow cells (Fig. 4B). Unlike DC fromthe peritoneal cavity, BM CD11c+ cells were DR– andDQ–. Upon culture in the presence of GM-CSF, approxi-mately 60% of CD11c+Mac-1lo cells became DR+ and afurther increase in percentage (70–80%) and intensity

(MFI 471–1,286) of DR expression was observed inmature DC upon LPS treatment. To our surprise, noinduction of DQ expression was seen in these cells prioror post-cytokine/LPS treatment (data not shown),although its appearance on peritoneal CD11c+ cells wasclearly identifiable. It is possible that the regulation of DQexpression is different from DR and the in vitro matura-tion process may not fully represent the in vivo differenti-ation milieu. Overall, we have demonstrated that matureDC in transgenic mice are capable of expressing DR andDQ and the expression is likely coupled with DC differen-tiation and maturation processes.

The identical expression pattern of HLA DR and DQ in4D1/C2D mice as compared to human cells indicatesthat the transgene construct contains most, if not all,necessary control elements that confer cell-specific reg-ulation of gene expression. It also suggests that the tran-scription regulation of the transgene is not influenced bythe sequences flanking the integration site.

The expression of HLA DR can be further regulated byvarious stimuli in APC and T cells. To understandwhether the same activation signals control DR geneexpression in transgenic murine cells, DR expressionwas compared in splenocytes before and after LPS oranti-CD3/anti-CD28 mAb stimulation. Up-regulation of


Fig. 3. Expression of DR3 and DQ2 molecules on B and T lymphocytes. (A) Transgene expression was analyzed by co-stainingLN cells with CD3, B220, and DR or DQ antibodies. Data shown are a representative of more than six experiments. As a compari-son, expression of DR and DQ in human B (CD19+) and T (CD3+) cells was performed. Percentage of DR+ and DQ+ cells in totalB or T cell population and the MFI of total B or T cell population are indicated. The results are summarized in histogram plots;where the thin lines indicate B6 control, gray lines 4D1/C2D, and dark lines human cells. (B) Correlation of memory phenotypeand DR expression in T cells was examined by staining cells with CD4 or CD8, CD44 and DR § antibodies. Events shown in dotplots are gated on CD4+ or CD8+ T cells that are either CD44– or CD44+.

DR expression in B cells upon LPS treatment was shownby an eight- to tenfold increase in MFI, whereas that in Tcells was demonstrated by both a threefold increase inDR+ cells ( ˚ 13% to ˚ 40%) as well as a two- to fourfoldincrease in MFI (Fig. 5A). Further characterization of theT cells revealed that both CD4 and CD8 T cells up-regulated DR to a similar degree, and the results werecomparable to that of human T cells (Fig. 5B). The shift of

non-T cell fraction towards higher levels of DR expres-sion upon anti-CD3/CD28 stimulation was most likelythe effect of IFN- + secreted by activated T cells on Bcells. The data clearly show that the large fragment ofhuman genomic DNA is not only able to confer cell-specific expression of DR and DQ genes it also containscis-regulatory elements for inducible expression in thesame fashion as seen in human cells.


Fig. 4. DC from transgenic mouse express DR3 and DQ2. (A)Peritoneal lavaged cells from B6 (gray) and 4D1/C2D (black)mice were stained with CD11c and DR or DQ antibodies.Human mature DC derived from in vitro cultured PBMC wereincluded as a comparison. In these plots, gray curves areisotype controls and black curves are human DC. (B) BMcells freshly isolated, cultured in the presence of GM-CSF, orcultured in the GM-CSF and stimulated with LPS werestained with antibodies against CD11c, Mac-1, and DR orDQ. Cells shown in history plots were gated on CD11c+ andMac-1lo. Numbers in the plots indicate MFI of DR expressionin DC from B6 (gray curves) and 4D1/C2D mice (blackcurves).

2.3 Thymic selection in transgenic mice

The formation of interspecies DR § /I-E g dimers has beenreported in DR § -only transgenic mice [26]. Our 4D1/C2Dmice, although in the I-A g –/– background, express I-E gmolecules intracellularly. To examine the presence ofDR § /IE g heterodimer formation in these mice, B lympho-cytes were stained with antibodies recognizing DR § ,DR g and IE g molecules. Among the DR § + population,80–93% were DR g + and G 90% were IE g + (Fig. 6A). Inagreement with the findings from a similar HLA trans-genic line [27], these data indicate that DR § /DR g andDR § /IE g are co-expressed on the cell surface. We alsoaddressed the possibility of I-A § /DQ g dimer formation.Although C57BL/6 and 4D1 transgenic mice in a I-A g +

background showed high levels of I-A § expressionwithin the B cell population, 4D1/C2D mice were identi-cal to C2D control mice and showed no detectable I-A §expression (Fig. 6B).

The presence of mature CD4+ T cells in the periphery atlevels equivalent to B6 mice suggests that the DR3 andDQ2 transgenes in the absence of mouse MHC class IImolecules (I-A § g and I-E § g ) are able to mediate thymicselection and peripheral homeostasis. To investigatehow well HLA molecules could generate diverse TCRrepertoire, T cells from thymus, spleen and LN were ana-lyzed with a panel of TCR V g antibodies. LN T cells from4D1/C2D mice contained clones that expressed most ofthe V g examined at levels comparable to B6 mice. Inter-estingly, V g 5-, V g 11- and V g 12-expressing cells wereunder-represented or absent from the transgenic mice inboth CD4 and CD8 populations. The same deletion wasalso observed in B10.A mice that express I-E molecules,as well as in the CD4 and CD8 single-positive thymo-cytes from both strains (Fig. 7 and data not shown). Inaddition, under-representation of V g 4 and V g 7 in CD4+ Tcells and V g 7, V g 9 and V g 14 in CD8+ T cells were almostidentical between 4D1/C2D and B10.A mice. However,changes unique to 4D1/C2D mice were identified as well.These included the decrease in the percentages of V g 3and V g 17 in both populations and V g 14 in CD4+ T cells.In addition to under-utilization of certain TCR clones,V g 6- and V g 10-expressing clones were preferentiallyselected in both 4D1/C2D and B10.A mice, with slightlyhigher representation in the former. More importantly,higher percentages of V g 8-expressing populations werefound in 4D1/C2D but not B10.A animals. Consistentwith the V g distribution of mature T cells, similar profilesof positive selection of V g 6, V g 8 and V g 10 were also evi-dent in the mature CD4 and CD8 single-positive thymo-cytes (data not shown). These results demonstrate thatHLA DR3 and DQ2 molecules are able to shape TCR rep-ertoire by mediating positive and negative selection pro-cesses during thymocyte development and by maintain-ing T cell peripheral homeostasis.

2.4 Functional analyses of cellular and humoralimmunity

To understand whether DR § /DR g dimers are functionallycompetent in triggering immune response, 4D1/C2Dmice were examined for their ability to process and pre-sent a known HLA DR3-restricted peptide epitope froman intact protein. Mice were immunized with CFA inwhich the Mycobacterium tuberculosis heat shock pro-tein (hsp) 65 contains an HLA DR3-restricted CD4+ T cellepitope at amino acid position 3–13 [28]. As a compari-son, mice were immunized with the hsp 1–20 peptide ineither CFA or IFA. As shown in Fig. 8A, LN cells from4D1/C2D mice immunized with CFA are capable ofmounting a proliferative response to the DR3-restrictedpeptide derived from the protein present in the immuno-gen. In contrast, proliferative responses to the peptide in


Fig. 5. Up-regulation of DR expression in activated B and T cells. Splenocytes from one B6 and two 4D1/C2D transgenic micewere cultured in the presence of LPS or anti-CD3/anti-CD28 mAb for 24 h and expression of DR was analyzed and comparedwith freshly isolated cells. Numbers indicate the percentages of DR+ cells in total B or T cells (A) and in total CD4 or CD8 T cells(B). Data are representative of three experiments.

Fig. 6. DR § forms dimer with both DR g and IE g , and I-A §does not dimerize with DQ g and DR g . (A) Total white bloodcells from peripheral blood were analyzed for DR § , DR g andIE g expression with specific antibodies. Cells were gated onthe B220+ population. Light lines represent B6 control anddark lines 4D1/C2D mice. (B) Dimerization and surfaceexpression of I-A § and DQ g or DR g were examined in trans-genic mice. LN cells from B6, 4D1/I-A g +, C2D and 4D1/C2Dmice were stained with B220 and I-Ab § . Data are representa-tive of at least four experiments.

IFA and in CFA were twofold higher than with CFA aloneas a result of an increased overall concentration ofimmunizing peptide. The slightly reduced response withCFA + hsp 1–20 was not statistically significant and likelydue to the saturating amount of hsp 1–20 (100 ? g/ml)used in both reactions that may plateau the responsesand mask the potential quantitative difference by usingdifferent adjuvant. We conclude that the HLA-DR3 mole-cules present in the 4D1/C2D mouse enter into the classII presentation pathway and bind peptides of the appro-priate specificity in conjunction with mouse processingintermediates.

To determine if 4D1/C2D mice are capable of respondingto protein antigen, we investigated antibody responsesto OVA. Groups of mice were immunized with OVA in aprotocol designed to induce high levels of antigen-specific antibody, most notably IgE. As shown in Table 1,4D1/C2D mice produce robust levels of IgG1, IgG2b andIgE on the order of the levels expressed by BALB/c mice.Neither BALB/c nor 4D1/C2D mice produced significantlevels of OVA-specific IgG2a. As expected, antigen-specific antibody production from control C2D animalsremained at background levels in separate experimentsperformed in an identical manner as well as in studies byothers (data not shown and [24]). This suggests thatantigen-primed CD4+ T cells from 4D1/C2D mice arecapable of cognate interactions with antigen-specific Bcells that result in isotype switching.


Fig. 7. TCR repertoire selection in 4D1/C2D mice. Cells from LN of 4D1/C2D, B6 and B10.A animals were examined for V g usageby flow cytometry. Percentage of V g -specific cells was quantified in CD4+CD3+ (upper panel) or CD8+CD3+ (lower panel) gatedpopulations. Results are graphed from a mean of three animals in each group and error bars indicate standard deviations. Datashown are results from one of two experiments.

Table 1. Antibody response of 4D1/C2D mice to immunization with OVAa)

Polyclonal IgG IgG1 IgG2a IgG2b IgE

BALB/c G 12 G 10 1.12±0.97 15.63±1.23 11.45±1.09

4D1/C2D G 12 G 10 0.90±1.57 G 10 11.07±1.80

a) Log2 titers of OVA-specific antibody are reported as averages for three mice. If a linear regression could not be performed thedata represented as a value G the maximum dilution tested.

2.5 T cells from 4D1/C2D mice respond toantigen presented by human APC

To further evaluate whether cells from 4D1/C2D miceindeed recognized antigen complexed to human DR3and DQ2, an antigen-presentation assay was performedwith human APC. CD4+ T cells from OVA-immunizedmice were cultured with OVA and human B-lymphoblastoid cells that are DR3+DQ2+ (8.1.6 cells) orDR null and DQ2+ (9.22.3 cells). A typical dose-dependent proliferative response to increasing amountof OVA was observed (Fig. 8B). Thus, T cells from immu-nized 4D1/C2D mice are proficient at recognizing andresponding to antigen presented by human HLA-DR3+DQ2+ APC, suggesting that they have beenselected against the right HLA haplotype in vivo.

3 Discussion

Here we describe phenotypic characterization of HLADR3/DQ2 transgenic mice generated utilizing a 550-kbYAC construct containing the complete DR § , DR g 1,DR g 3, DQ § , and DQ g loci. A large genomic DNA frag-ment (4D1) used in this study has a greater potential tomimic human physiological conditions and provide thecell-specific distribution and activation-dependent up-regulation of class II molecules seen in humans. Indeed,our data show the restricted expression of DR and DQ inB cells, DC and a low percentage of T cells, and the up-regulation of DR in B and T cells. One aspect of differen-tial regulation of class II genes in mouse and human isthe absence of class II in murine T cells [29]. Unlike mostHLA class II transgenic mice, our transgenic mouse with


Fig. 8. 4D1/C2D mice process and present HLA transgene-restricted epitopes and respond to antigen in the context of DR3 andDQ2. (A) Draining LN were removed from mice immunized with CFA, IFA + 100 ? g hsp 1–20 peptide, or CFA + 100 ? g hsp 1–20peptide and tested in vitro with 10 ? g/well hsp 1–20 peptide. ¿ CPM was determined for each animal, and the results were aver-aged. (B) CD4+ T cells from OVA-immunized mice were co-cultured with human B-LCL and OVA. T cell stimulation was measuredby [3H]thymidine uptake. Data were plotted as an average of triplicates. This experiment was performed three times with similarresults.

cognate cis-control elements showed low levels of DRexpression in ˚ 12–18% of resting T cells and ˚ 40% ofactivated T cells. This suggests the presence of uniquecis-acting regulatory sequences in the human class IIlocus that confer cell type- as well as activation-dependent gene expression. Other transgenic mice cre-ated with a large genomic construct encompassing bothHLA-DR3/DQ2 did not show DR expression in resting oractivated T lineage cells [27]. This is likely due to thesmaller size of the construct (320 kb). Alternatively, theother strain of mice may carry their transgene in a loca-tion that is subject to flanking-region effects.

The inclusion of DR and DQ present in their natural link-age positions in the transgenic construct allows compar-isons to single DR and DQ transgenic mice. Studiesalong this line have shown that, whereas DR3 mediatesthe onset and susceptibility of the disease, DQ8 modifiesits severity [30]. Separate studies have shown that theDR2 molecule modulates collagen-induced arthritis inassociation with the expression of DQ8, and DR4reduces the incidence of spontaneous diabetes in DR4/DQ8/RIP-B7 transgenic mice [2, 31]. Events that mayattribute to disease modification include: (1) epitopemasking and epitope spreading in autoantigens, eitherallowing certain autoreactive T cells to remain ignorant orresulting in recruitment and expansion of T cells withadditional autoantigen specificities, respectively [13, 30];(2) switching of the CD4 T cell response from Th1 to Th2;and (3) reshaping the TCR repertoire [2, 18, 31]. Thesestudies elegantly demonstrate the interplay between dif-ferent DR and DQ alleles in mediating autoimmunity anddemonstrate the usefulness of HLA transgenic mice inunderstanding disease pathogenesis and immune regu-lation. However, all of these double transgenic modelscarry two discrete transgenes that are not linked as in

their naturally occurring configuration, with control ele-ments and potential loci-associated genes in atten-dance. It will be interesting to evaluate these studies ingenomic HLA loci transgenic mice such as 4D1/C2D.

The role of murine I-E molecules in modulating diseaseprocesses has been demonstrated in several differentautoimmune disease models. For instance, expressionof I-E renders resistant strains susceptible for autoim-mune thyroiditis [32] and confers resistance to diabetesin NOD mice [33, 34]. DR § /I-E g dimers have beenreported to be functionally indistinguishable from I-E § g[26]. It has also been demonstrated that DR § /DR g , DR § /I-E g and I-E § /I-E g dimers select TCR repertoire with sim-ilar V g usage [26, 35, 36]. Although our 4D1/C2D micedid show near complete deletion of V g 5, V g 11 and V g 12cells and partial deletion or under-representation of a fewother V g that are consistent with I-E-expressing mice,reduced utilization of V g 3, V g 14 and V g 17 and over-representation of V g clones that are unique to 4D1/C2Dmice suggest the participation of DR3, DR g 52 and DQ2molecules in the selection process. The combination ofmultiple diverse class II molecules confers a unique pat-tern of TCR repertoire. Interestingly, others havereported that trans-species dimerization of DR § /I-E g isless efficient than DR § /DR g in triggering antigen-specificT cell activation and that less than ˚ 5% of antigen-specific T cell hybridomas raised in DR4 transgenic miceare restricted to DR § /I-E g [15]. This observation corrob-orates findings made by us and others that despite thepresence of DR § /I-E g dimer, APC in the transgenic miceare able to process and present a DR3-restricted antigenand induce an antigen/DR3-dependent T cell response[37]. These observations suggest that the 4D1/C2Dtransgenic mice in their current genetic background canbe used to investigate HLA-related immunological ques-


tions. To unequivocally resolve the I-E g interference,breeding DQ2/DR3 transgenic mice to I-A/E doubleknockout background is required [38].

In conclusion, we demonstrate the importance of using alarge fragment of human genomic DNA to confer cell-specific expression of DR and DQ genes and its ability toprovide cis-regulatory elements for inducible expressionin the same fashion as seen in human cells. We alsoshow that the HLA-DR3 and -DQ present in the 4D1/C2Dmouse enter into the class II presentation pathway andbind peptides of the appropriate specificity in conjunc-tion with mouse processing intermediates and thatantigen-primed CD4+ T cells from 4D1/C2D mice arecapable of cognate interactions with antigen-specific Bcells that result in isotype switching. We anticipate thatthe 4D1/C2D mouse will serve as a relevant diseasemodel and allow for more reliable target identificationand validation for immunotherapies.

4 Materials and methods

4.1 Generation of transgenic mouse

A 550-kb human genomic fragment was isolated as a YAC(clone 4D1) and used to generate transgenic mice. It encom-passes a region of human chromosome 6 that contains theTAP1/2, LMP2/7 and HLA DQw2 (DQA*0501/DQB1*00201)and DR3 (DRA*0101/DRB1*0301/DRB3*52) genes (Fig. 1)[21]. The YAC was co-transfected into AB-1 embryonic stemcells [39] with a plasmid carrying the phosphoglyceratekinase promoter-driven neor gene [40] using transfectam(Promega, Madison, WI). G418-resistant clones werescreened for transgene integration by PCR and Southernblotting analyses. Embryonic stem cells identified as con-taining at least one intact copy of the 4D1 YAC were injectedinto blastocysts derived from C57BL/6 mice. Offspringderived from transgenic ES cells were screened by tail DNAanalysis and transgene positive founders were crossed toC57BL/6 mice and to MHC class II I-A g –/– mice (Taconic,Germantown, NY) [24].

4.2 Cells

Lymphocytes were obtained from thymus, spleen and LN of4D1/C2D and B6 mice. Mature DC were obtained by perito-neal lavage. BM-derived immature and mature DC weregenerated as described [41]. Briefly, bone marrow cells fromB6 and 4D1/C2D mice were cultured with 20 ng/ml GM-CSF(Endogen, Inc. Woburn, MA) for 10 days followed by 1 ? g/mlLPS (Sigma) for 1 day. CD11c+Mac-1lo cells were analyzedfor DR and DQ expression. Human PBMC were preparedfrom buffy coat material (Stanford University Blood Center).Mature human DC were derived as previously described[42]. Human B lymphoblastoid cell line (B-LCL) 8.1.6 cells

expressing DRB1*0301, DRw52, DQ2 and DP4, and itsderivative 9.22.3 cells lacking DR § and expressing DQ2 andDP4 were a kind gift from Dr. E. D. Mellins (Stanford Univer-sity) [43].

4.3 Phenotypic analysis

Cells were stained with phycoerythrin (PE), fluorescein iso-thiocyanate (FITC), or allophycocyanin-conjugated anti-bodies that recognize B220, CD3, CD4, CD8, CD19, CD44,CD11c, I-E g (clone 17-3-3), I-Ab § (clone AF6-120.1), HLADR (clones L243 and TÜ36), and DQ (clone 1a3). TCR reper-toire was analyzed using a panel of V g antibodies. All anti-bodies used in this study were purchased from BD PharMin-gen (San Jose, CA), except anti-DQ antibody (BiodesignInternational, Saco, ME). Flow cytometry was performedusing FACSCalibur and CellQuest software (Becton Dickin-son, San Jose, CA).

4.4 Functional analysis

Mycobacterium tuberculosis hsp65 peptide 1-20 (MAKTIAY-DEEARRGLERGLN) was constructed in house using Fast-Moc chemistry with the ABI peptide synthesizer. Mice wereimmunized in their hind footpads, and popliteal LN wereremoved 7 days later. LN cells were cultured with 10 ? g/ wellof the peptide for 24 h. Proliferation was assessed 18 h later[28].

OVA-specific IgG1 and IgE responses were induced asdescribed [44]. OVA-specific antibodies were measured byELISA using biotinylated anti-IgG1 (A85-1), anti-IgE (R35-118), anti-IgG2b (R12-3), horseradish peroxidase-conjugated anti-IgG2a (R19-15) (all from PharMingen), andanti-IgG Fc-specific antibodies (Jackson Immunoresearch).

The mouse T cell response to antigen presented by humanAPC was performed as follows. Mice were immunized asdescribed above; with schedule modifications of intraperito-neal immunization (day 1) followed by intranasal immuniza-tion on days 7, 8 and 9. Mice were killed on day 10. CD4+ Tcells purified from spleen and LN using Miltenyi beads (Mil-tenyi Biotec, CA) were cultured with B-LCL 8.1.6 or 9.22.3cells pretreated with mitomycin C (Sigma-Aldrich, MO) in thepresence of increasing concentrations of OVA (WorthingtonBiochemical, NJ) for 72 h at 37°C. Cells were pulsed with[3H]thymidine and proliferation was assessed.

Acknowledgements: The authors thank Jiannis Ragoussisfor the 4D1 YAC construct, Dr. E. D. Mellins for human Blymphoblastoid cell lines, Doug Hodges for mouse geno-typic analysis, Regina Chin and Nargol Faravashi for assis-tance in proliferation assays, and Jim McCabe and DanielBrigham for animal facility management and assistance insurgical procedures. We also thank Dr. Mark Goldsmith and


members of the Immunology group for helpful discussionand comments, and Roopa Ghirnikar for her editorial assis-tance. This work was partially supported by the NationalInstitute of Standards and Technology, Advanced Technol-ogy Program, Cooperate Agreement no. 70NAB4H151.

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Correspondence: Dan Chen, Genencor International Inc.,925 Page Mill Road, Palo Alto, CA 94304, USAFax: +1-650-621-7826e-mail: dchen — genencor.com


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Characterization of HLA DR3/DQ2 transgenic mice: a potential humanized animal model for autoimmune disease studies